Bidirectional Regulation of Endothelial Nitric Oxide Synthase Phosphorylation and Function by Reactive Oxygen Species

Abstract

Phosphorylation of eNOS Ser1179 by Akt is a central mechanism of eNOS regulation. Hydrogen peroxide (H2O2) was reported to enhance eNOS Ser1179 phosphorylation function. But prior studies were performed on serum-starved cells and only the transient effect of H2O2 was determined. These results appear to be in paradox with the evidence that oxidants decrease eNOS function in chronic diseases. Here we found that the effects of H2O2 on eNOS phosphorylation and function are bidirectional. Our experiments were conducted on BAECs or bovine eNOS stably transfected cells cultured with serum (10% FBS). H2O2 (1–500 uM) initially upregulated eNOS phosphorylation. The phosphorylation of eNOS Ser1179 was increased 2-fold at 30 min of H2O2 treatment. Accordingly, phospho-eNOS activity was elevated (19.7 ± 2.5 vs 41.8 ± 2.5 pmol/mg/min, P<0.01, n=5). However, after the peak increase at 30 min, eNOS Ser1179 phosphorylation dramatically declined. At 8 hr of H2O2 treatment, the levels of Ser1179-phoshporylated eNOS were reduced more than 3-fold and phospho-eNOS activity was largely diminished. This bidirectional action was specific because the phosphorylation status of eNOS Thr497 was unchanged. Parallel to the alterations of eNOS Ser1179 phosphorylation, a synchronic increase and decline of active Akt were seen. Blockade of Akt activation by PI3K inhibition largely prevented H2O2-induced eNOS Ser1179 phosphorylation. Pull-down assays showed that H2O2 did not disrupt eNOS-Akt association in cells. These studies revealed a crucial bidirectional action of H2O2 on eNOS Ser1179 phosphorylation and activity. Opposite to its initial activating effect, long-term H2O2 exposure deactivated Akt and reduced eNOS Ser1179 phosphorylation and function.